Study On The Effect And Mechanism Of PHB1 In The Regulation Of Glucose Metabolism Through TFAM In Nasopharyngeal Carcinoma Cells

Purpose:To clarify the effect of anti-proliferative protein(Prohibitin1,PHB1)on the proliferation and glucose metabolism of nasopharyngeal carcinoma(NPC)cells,and to explore the molecular mechanism of PHB regulating the glucose metabolism of NPC cells through mitochondrial transcription factor A(TFAM,TCF6).Methods:1.Plate CCK-8 cell proliferation assay,glucose uptake assay,extracellular lactate secretion assay,G6 PD activity assay and G6 P activity assay were used to analyze the effects of expressing PHB1 on the proliferation and glucose metabolism of nasopharyngeal carcinoma.Bioinformatics analysis,Western Blot and Mito-tracker Red immunofluorescence assay were used to analyze the effects of PHB1 on the expression of glucose metabolism,mitochondrial membrane potential,and mitochondrial function-related molecules.Results:1.CCK-8 assay showed that PHB1 inhibited the proliferation of nasopharyngeal carcinoma cells in vitro;we used glucose uptake assay,extracellular lactate secretion assay and G6P/G6 PD activity assay to analyze the regulation of glucose metabolism in nasopharyngeal carcinoma cells,and the results showed that PHB1 inhibited glucose uptake,extracellular lactate secretion and G6P/G6 PD activity in nasopharyngeal carcinoma cells.2.Western blot assay showed that PHB1 inhibited the expression of glycolytic rate-limiting enzyme pyruvate kinase(PKM2),lactate dehydrogenase A(LDHA)and pentose phosphate pathway rate-limiting enzyme glucose-6-phosphate dehydrogenase(G6PD),and promoted the expression of tricarboxylic acid cycle metabolizing enzymesα-ketoglutarate OGDH,aconitase ACO2 and succinate dehydrogenase SDHB in nasopharyngeal carcinoma cells.The results of bioinformatics analysis showed that the expression of tricarboxylic acid cycle enzymeα-ketoglutarate OGDH,aconitase ACO2 and succinate dehydrogenase SDHB was enhanced.3.Bioinformatics analysis showed that TFAM was lowly expressed in nasopharyngeal carcinoma cells and nasopharyngeal carcinoma tissue samples,and PHB1 had a positive correlation with TFAM;Western blot assay showed that PHB1 enhanced the expression of TFAM in nasopharyngeal carcinoma cells;protein interaction network analysis revealed that TFAM had a strong protein interaction with PHB1,and had a strong protein interaction with mitochondrial structure,function and glucose metabolism molecules.The protein interaction network analysis revealed that TFAM has strong protein interaction with PHB1 and interactions with mitochondrial structure,function and sugar metabolism molecules.4.We used mitochondrial immunofluorescence assay to find that the mitochondrial structure and mitochondrial membrane potential were significantly improved and enhanced after overexpression of PHB1,while silencing the expression of TFAM after overexpression of PHB1,the mitochondrial structure and mitochondrial membrane potential returned to the NC group level;we analyzed the effect of PHB1 and TFAM on mitochondrial structure and function related molecules by Western blot assay.Detection,PHB1 was found to promote the expression of mitochondrial folding protein HSP60 and mitochondrial membrane dynamics OPA1,DRP1,FIS1,Mfn1,Mfn2 through TFAM.5.The glucose uptake assay,extracellular lactate secretion assay,and G6P/G6 PD activity assay methods revealed that the glucose uptake assay,extracellular lactate secretion assay,and G6P/G6 PD activity of the experimental group overexpressing PHB1 but silencing TFAM were all downregulated compared with the PHB1 overexpression control group;CCK-8 assay showed that the proliferation rate of the experimental group overexpressing PHB1 while silencing TFAM expression was increased compared with the PHB1 overexpression control group.Conclusion:1.PHB1 inhibit the proliferation of nasopharyngeal carcinoma cells in vitro.2.PHB1 is participates in the regulation of carbohydrate metabolism in nasopharyngeal carcinoma cells,which may be related to the maintenance of mitochondrial membrane potential and function by PHB1 through the mediation of TFAM.